Interpretive Summary: Poly(hydroxyalkanoates) (PHAs) are biodegradable plastics synthesized by many bacteria and have great potential for use as ecologically sound substitutes for petroleum-based polymers in the fabrication or formulation of biomedical devices, consumer products, packaging materials, and adhesives. Although PHAs are useful, they are expensive to produce and lack good materials properties. These challenges can potentially be overcome by genetically modifying the producing bacteria to change their metabolic properties in a process called metabolic engineering. The understanding of the structure-function relationship of the bacteria’s PHA-biosynthesis genes is important to perform metabolic engineering of the microorganisms. In this paper, we report the complete characterization of the gene sequence responsible for PHA synthesis in a bacterium called Pseudomonas corrugata. Based on the results of an extensive sequence characterization, we constructed two mutants of P. corrugata that lack a unique DNA structure called an intrinsic terminator. We showed that these mutants when grown on glucose produced PHA with a special composition that allows for subsequent chemical modification if desired. We also showed that the shape of the PHA granules produced by these mutants grown on a fatty acid called oleic acid are different from those found in the wild-type (i.e., non-genetically engineered) cells. These results not only describe for the first time the structure-function relationship of an intrinsic terminator in relation to PHA synthesis, but also provide a means to produce highly reactive PHA for further chemical modification. Further exploitation of these results should lead to processes to make new PHA for use in various applications to benefit the consumer.

Technical Abstract:
The complete sequence of the pha locus responsible for the biosynthesis of poly(hydroxyalkanoates) (PHAs) in Pseudomonas corrugata 388 was determined. As with the other known pseudomonad pha gene loci, the one in P. corrugata 388 also consists of phaC1 (1680 bps; PHA synthase 1), phaZ (858 bps; PHA depolymerase) and phaC2 (1683 bps; PHA synthase 2) genes. A BLAST search showed that the nucleotide sequences of these genes and the amino-acid sequences of their respective gene products are homologous to those of P.corrugata CFBP5454 and P. mediterranea CFBP5447. A putative intrinsic transcription terminator consisting of a dyad symmetry (24 bps; Delta G = -41.8 kcals) that precedes a stretch of dA residues was located in the phaC1-phaZ intergenic region. P. corrugata mutant-clones XI 32-1 and XI 32-4 were constructed in which this intergenic region was replaced with a selectable kanamycin-resistance gene. In comparison to parental P. corrugata with only a few large PHA inclusion bodies, the mutants grown on oleic acid produce numerous smaller PHA granules that line the peripheral of the cells. With glucose as a substrate, XI-32-1 and XI 32-4 clones produce mcl-PHA with a high content (32-33 mol%) of the mono-unsaturated 3-hydroxydodecenoate as a repeat-unit monomer. Our results show for the first time the effects of the phaC1-phaZ intergenic region on the repeat-unit composition of mcl-PHA and the morphology of the PHA granules.